This proposal will investigate the potential for B cell immunoregulation by CD180 stimulation and study the mechanisms of CD180 interactions with BCR and TLR signals. CD180 (RP105) expression is required for TLR4 and TLR2-mediated B cell proliferation and T cell- independent antibody responses (TI-1 response). We propose a comprehensive study of the CD180 receptor, including studies of the mechanisms of CD180 interaction with BCR and TLR signals, combined with analysis of CD180 activation in mice to determine the effect on antigen-specific B cells expressing transgenic high affinity BCR (B1-8hi) specific for NP, and on the antibody response to NP-CGG. These experiments will determine whether CD180 activation may serve as a sensitization signal that could result in selective B cell deletion upon antigen binding. CD180 is thought to coordinate or integrate the TLR4 and TLR2 signals with BCR signals in B cells to drive an antigen specific antibody response of all isotypes. We have found that CD180 stimulation together with TLR agonists results in synergistic B cell proliferation, and augments B cell production of inflammatory cytokines IL-6, IL-10, and TNF1. In contrast, proliferation of CD180-stimulated B cells is inhibited by simultaneous BCR stimulation, and CD180-activated B cells are sensitized to anti-BCR apoptosis. The second major component of this grant will use molecular engineering of soluble forms of CD180/MD-1 that are expressed in mammalian cells to help identify and potentially block natural CD180 ligands. We will also express anti-human and anti-mouse CD180 scFv-Ig fusion proteins. The scFvs will be constructed with wt or mutated human and mouse Ig tails in order to study the regulation of CD180 signals by FcR binding. The results will help clarify the role of CD180 as a master regulator of TLR2 and TLR4 and will help determine the potential for CD180 as a target for antibody-based therapy. The work will also result in construction and expression of recombinant molecules that will be useful in future studies of CD180 therapy in infectious disease and cancer models. PUBLIC HEALTH RELEVANCE: CD180 is a critical receptor for regulation of TLR4 and TLR2 function in B cells and could be an important target for immunoregulation. This grant will express recombinant molecules for CD180 stimulation and will test the potential for CD180 as a therapeutic target for deletion of autoreactive B cells.